This proposal examines the regulation of sonic hedgehog (Shh) response in developing cerebellum. Sonic hedgehog produces multiple morphogenetic and mitogenic effects during development including cerebellar granule cell proliferation. Dysregulation of Shh has profound developmental consequences and is implicated in several cancers. The mechanism of Shh signaling is poorly understood and lags far behind our knowledge of several other growth factors. In Drosophila, Tout-velu, a putative glycosyltransferase involved in the synthesis of heparin sulfate proteoglycans (HSPG) is necessary for Hh response. There are multiple examples of HSPG modulation of growth factor signaling and several diseases of HSPG synthesis with significant developmental abnormalities and increased cancer risks. We have focused on the effects of HSPGs on Shh receptor binding and signaling in the developing mouse cerebellum. In preliminary studies we have demonstrated direct interaction of Shh with heparin, and shown that reduction of cell surface HSPGs results in decreased Shh induced proliferation consistent with the effect of the Tout-velu mutation. Surprisingly, these same treatments result in increased Shh binding. Further, we have found that cerebellar expression of EXT2, a vertebrate Tout-velu homolog, increases with age and is more abundant in post-mitotic granule cells. We hypothesize that the synthesis of HSPGs is regulated during cerebellar development and is a determinant of Shh binding and Shh responses. We will test our hypotheses by determining the pattern of expression of EXT family members (EXT1-3, L1-3) in developing cerebellum. We will measure Shh pathway activation in primary cultures of cerebellum from Ptc-LacZ (plus/minus) mouse and Shh receptor binding after genetic and biochemical manipulation of HSPG synthesis. The necessity for direct HSPG-Shh interactions will be determined by site-directed mutagenesis of the putative Shh heparin binding domain. Taken together these experiments will generate novel information regarding HSPG modulation of the relationship between Shh binding and signaling and may shed new light on the mechanisms by which dysfunction of Shh and HSPG produce disease. In addition, these studies will provide me with the opportunity to learn the techniques of modern cell and molecular biology and establish the skills with which to study neural developmental. Dr. Rosalind Segal's laboratory in the Department of Pediatric Oncology at the Dana-Farber Cancer Institute provides an excellent environment for me to develop as an independent investigator.